P1256: A NOVEL DROP-OFF DIGITAL PCR ASSAY FOR CXCR4 MUTATION SCREENING IN IGM GAMMOPATHIES: FIRST DATA FROM THE FONDAZIONE ITALIANA LINFOMI BIO-WM STUDY
Authors: D. Drandi, M. Ferrante, S. Zibellini, L. Marcheselli, E. Cappello, M. Borriero, S. Ragaini, I. Dogliotti, C. Varraso, F. Cavallo, A. Ferrari, M. Merli, G. Zamprogna, L. Laurenti, S. Tomasetti, E. Cencini, G. Loseto, S. Finotto, M. Marchetti, F. Re, A. Sica, J. Olivieri, C. Jimenez, N. Puig, C. Cavalloni, R. García-Sanz, M. Varettoni, S. Ferrero
Journal: HemaSphere (2022)

Background: In IgM gammopathies: Waldenström’s Macroglobulinemia (WM) and IgM gammopathy of uncertain significance (IgM-MGUS), MYD88L265P and CXCR4S338X are the most frequent mutations, and both have clinical implications in WM. Recent studies demonstrated that, in unselected bone marrow (BM) samples, allele specific polymerase chain reaction (AS-qPCR) was superior in detecting CXCR4S338X compared to next generation sequencing, and digital PCR (dPCR) was more sensitive than AS-qPCR for MYD88L265P detection, highlighting the lack of a gold standard molecular method in WM diagnostics. Aims: The aim of the present study was to evaluate a new sensitive approach based on a drop-off dPCR assay for CXCR4S338X mutation detection in BM and peripheral blood (PB) samples from WM and IgM-MGUS patients, enrolled between 2018 and 2020 in the multicenter observational trial from the fondazione italiana linfomi (“BIO-WM”, NCT03521516), and to correlate the mutational status with clinical features. Methods: Genomic DNA from 240 patients (184 WM, 56 IgM-MGUS) was analyzed by dPCR: 51 BM-CD19 selected cells (CD19+), 189 BM and 239 PB unselected white blood cells (WBC). Allele frequency (AF) of CXCR4 mutations at p.S388 locus (CXCR4MUT) was detected by a drop-off dPCR assay, with a sensitivity of 0,001%. For all patients, MYD88L265P mutation data were also available. Results: Forty-one out of 184 WM patients (22%) showed CXCR4MUT in BM (median AF 1.6%, range: 0.14%-23.1%) while only 8 were positive also in PB (20% of the BM mutated cases, median AF: 0.6%, range: 0.23%-35.2%). Moreover, 39/41 (95%) were MYD88L265P, too. Conversely, only 5 out of 56 IgM-MGUS patients (9%) scored CXCR4MUT in BM (median AF 0.65%, range: 1.1%-0.2%) and none in PB: all patients were MYD88L265P, too. Interestingly, 38 BM WBC samples (36 WM, 2 IgM-MGUS) mutated for both CXCR4MUT and MYD88L265P showed correlation in mutational levels between the two mutations (R2=0.8). On the contrary, no correlation was observed between the two mutations in CD19+ cells (R2=0.06). Overall, clinical features of CXCR4MUT vs CXCR4WT patients did not differ significantly, except for lower hemoglobin levels (median 11.6 g/dl vs 12.8 g/dl, p=0.002) and higher serum IgM monoclonal component (median 1.76 g/dl vs 1 g/dl, p=0.016). Only 15/41 WM CXCR4MUT patients (37%) received immediate treatment at the time of trial enrollment (7 DRC, 3 R-Benda, 5 others: 2 single agent and 3 R-chemo based therapy). These cases showed higher disease and mutational burden compared to the 26 CXCR4MUT patients still in watch and wait (WW) at the time of the present analysis. Indeed, median histologic BM infiltration was 80% vs 30% (p<0.001), median IgM monoclonal component was 3.6 g/dl vs 1.6 g/dl (p=0.003) and median CXCR4MUT AF was 9.5% vs 0.8% (p<0.001), respectively. Finally, at the present the median follow-up (FU) of the trial is relatively short (34 months) and no statistically significant differences in outcome parameters were recorded between CXCR4MUT and CXCR4WT cases. Summary/Conclusion: We here describe a sensitive, easily applicable and standardizable approach for detection of the most common CXCR4 mutations on BM WBC, with no need for CD19+ selection. A lower mutational AF was observed in IgM-MGUS compared to WM and, similarly to what was previously reported for MYD88L265P, PB resulted suboptimal for CXCR4MUT detection. A statistically significant difference in CXCR4MUT level was observed between patients treated at the time of enrollment and those still in WW. However, a longer FU is needed to better clarify the clinical implications of CXCR4 mutations in our series.